*[[Silver: Ligation|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.

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*[[Richard_Lab:Ligation]] -- Uses T4 Ligase

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*[[BE.109:DNA engineering/DNA ligation and bacterial transformation|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

[[Silver: Ligation|Silver:Ligation]] -- A protocol for sticky end ligations using the Roche Kit.

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-

+

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[[Richard_Lab:Ligation]] -- Uses T4 Ligase

+

-

+

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[[BE.109:DNA engineering/DNA ligation and bacterial transformation|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Curators

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. This is a new initiative on OWW, please provide your thoughts on the idea of consensus protocol curators here.

Abstract

DNA ligation is the process of joining together two DNA molecule ends (either from the same or different molecules). Specifically, it involves creating a phosphodiester bond bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. This reaction is usually catalyzed by a DNA ligase enzyme. This enzyme will ligate DNA fragments having blunt or overhanging, complementary, 'sticky' ends. Typically, it is easier to ligate molecules with complementary sticky ends than blunt ends. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.

The two components of the DNA in the ligation reaction should be equimolar and around 100μg/ml. Most commonly, one wants to ligate an insert DNA molecule into a plasmid, ready for bacterial transformation. Typically, DNA and plasmid vector are individually cut to yield complementary ends, then both are added to a ligation reaction to be circularised by DNA ligase. If the plasmid backbone to insert DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If the ratio is too low then the result may be an excess of linear and circular homo- and heteropolymers.

Procedure

10μL Ligation Mix

Calculating Insert Amount

The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.

Method

Add appropriate amount of deionized H2O to sterile 0.6 mL tube

Add 1 μL ligation buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.

Add appropriate amount of insert to the tube.

Add appropriate amount of vector to the tube.

Add 0.5 μL ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.

Using DNA fragments that have been exposed to UV during the gel extraction procedure (can avoid by blind excision, or by using a black-light or 365nm UV transilluminator instead of the usual 312nm type)

Using the NEB Quick Ligation Kit (heat inactivation of PEG in the buffer ruins transformation, without heat inactivation the ligation probably would've been fine)

For additional troublshooting, check out the NEB FAQ page for T4 ligation: [1]

Notes

Make sure the buffer is completely melted and dissolved. The white precipitate is BSA according to NEB. Make sure the buffer still smells strongly like "wet dog" (to check if the DTT is still good).

Because ligase buffer contains ATP, which is unstable and degraded by multiple freeze/thaw cycles, you may want to make 10-20ul aliquots from the original tube. Ligase buffer may be spiked with additional ATP.

Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.

Tom Knight has read that ligase can inhibit transformation [1]. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.